We will dissect the dendritic cell (DC) system in people to promote development of an HIV vaccine. As a result of hard fought progress in the last funding period, we wish to address 3 major obstacles and with a greater emphasis on monocytes and monocyte derived DCs (Mo-DCs). Aim 1 is to better identify and characterize DC and monocyte subsets in blood, stressing C-type lectin receptors (CLR) as markers as is done in mice, and their innate responses to adjuvants in vivo in healthy volunteers. Adjuvants for defined pattern recognition receptors are an essential part of DC-based protein vaccines, which we have developed to initiate strong T and B cell immunity in mice and monkeys and which have entered human studies. However we must overcome an obstacle to work on human DC and monocyte subsets in blood and lymphoid organs, because current markers are not based on known DC functions, are insufficiently specific, and most have not been studied in lymphoid tissues or in humans receiving vaccines and adjuvants. We will develop better functional markers for monocyte and DC subsets, emphasizing CLR for uptake and processing of both pathogens and vaccines and study their features in blood from healthy individuals who have or have not received adjuvants. Aim 2 will use hu-NSG (immunodeficient) mice to understand lymphoid tissue responses to adjuvants, both classical and Mo- DCs, and then protein vaccines targeted to CLR. With Christian M[unreadable]nz, it has been shown that these mice the major subsets of human DCs, organized T cell areas, and an ability to form HLA-restricted T cells. The gaps to overcome now are to use hu-NOG mice to identify DC and monocyte subset stressing: a) new criteria for classical and Mo-DCs 3, b) responses in organized lymphoid organs to adjuvants, and c) development of protein vaccines targeted to these subsets. Aim 3 will elucidate the Mo-DC pathway in vivo during responses to microbial mimics and vaccination. The gap we addressed in the past 5 years is to identify ways to identify and mobilize authentic Mo-DCs in vivo, i.e., monocyte-derived cells that localize to T cell areas and act as powerful antigen presenting cells. To do so, we developed workable antibodies to mouse DC-SIGN, which proved exceptionally difficult to obtain, but with these, we found that mouse DC-SIGN was an incisive marker for mouse Mo-DCs mobilized by TLR4 ligation by bacteria and cell walls. These Mo-DCs lose critical markers of inflammatory monocytes, and like DCs, localize to T cell areas. They are as or more active than classical DCs from the same lymph nodes, and are more numerous. We now aim to determine the role of human Mo- DCs in vivo in concert with aims 1 and 2, looking for Mo-DCs in volunteers during exposure to safe synthetic TLR4 agonists for humans.